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Hydrophobic microbeads as an alternative pseudo-affinity adsorbent for recombinant human interferon-α via hydrophobic interactions
Materials Science and Engineering C 32 (2012) 937–944
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Hydrophobic microbeads as an alternative pseudo-affinity adsorbent for recombinant human interferon-α via hydrophobic interactions Yeşeren Saylan a, Müfrettin Murat Sarı b,⁎, Serpil Özkara c, d, Lokman Uzun a,⁎, Adil Denizli a a
Hacettepe University, Department of Chemistry, Biochemistry Division, Ankara, Turkey Turkish Military Academy, Department of Basic Sciences, Ankara, Turkey Inonu University, Faculty of Pharmacy, Biochemistry Division, Malatya, Turkey d Anadolu University, Department of Chemistry, Biochemistry Division, Eskişehir, Turkey b c
a r t i c l e
i n f o
Article history: Received 15 January 2011 Received in revised form 15 November 2011 Accepted 2 February 2012 Available online 9 February 2012 Keywords: Recombinant human interferon-α Hydrophobic interaction chromatography Phenylalanine Poly(HEMA–MAPA) microbeads Affinity adsorption Pseudo-affinity adsorbents
a b s t r a c t Hydrophobic interaction chromatography (HIC) is increasingly used for protein purification, separation and other biochemical applications. The aim of this study was to prepare hydrophobic microbeads and to investigate their recombinant human interferon-α (rHuIFN-α) adsorption capability. For this purpose, we had synthesized functional monomer, N-methacryloyl-L-phenylalanine (MAPA), to provide a hydrophobic functionality to the adsorbent. The poly(2-hydroxyethyl methacrylate-N-methacryloyl-L-phenylalanine) [poly(HEMA–MAPA)] microbeads were prepared by suspension copolymerization. microbeads were characterized using FTIR, swelling behavior, and SEM micrographs. Equilibrium swelling ratio of poly(HEMA– MAPA) and poly(HEMA) microbeads were 53.3% and 69.3%, respectively. The specific surface area and average pore diameters determined by BET apparatus were 17.4 m2/g and 47.3 Å for poly(HEMA) microbeads and 18.7 m2/g and 49.8 Å for poly(HEMA–MAPA) microbeads. Adsorption experiments were performed under different conditions. Maximum rHuIFN-α adsorption capacity was found to be 137.6 ± 6.7 mg/g by using poly(HEMA–MAPA) microbeads with a size range of 150–250 μm and containing 327 μmol MAPA/g microbeads. Compared with poly(HEMA–MAPA) microbeads, nonspecific rHuIFN-α adsorption onto plain poly(HEMA) microbeads was very low, about 4.2 ± 2.3 mg/g. To determine the effects of adsorption conditions on possible conformational changes of rHuIFN-α structure, fluorescence spectrophotometry was employed. Repeated adsorption–elution processes showed that these microbeads are suitable for repeatable rHuIFN-α adsorption. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Interferons, named for its ability to interfere with viral proliferation, are very important proteins for the immune system and are the members of large class of glycoproteins known as cytokines. In some cases, they modulate specific cellular functions but the main function of these proteins is playing an important role in resistance to infection especially in the first line of defense against viral infections. They are part of the non-specific immune system and are induced at an early stage in viral infection before the specific immune system has had time to respond. Interferons show their main function as preventing viral replication in newly infected cells. These potent biologically active proteins are most rapidly produced by somatic cells in response to an appropriate stimulus in the presence of pathogens such as viruses, bacteria, or parasites, or other antigens or tumor cells. Then, they are secreted into the surrounding medium, bind to receptor on target cells and induce transcription of some genes and ⁎ Corresponding authors. Tel.: + 90 312 417 5190; fax: + 90 312 418 3226. E-mail addresses: [email protected] (M.M. Sarı), [email protected] (L. Uzun). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.02.016
these results in an anti-viral state in the target cells. Since interferons have different antigenic and structural characteristics, they are typically divided into three types: alpha (α), beta (β) and gamma (γ) [1]. Human interferon-α (HuIFN-α, also known as leukocyte interferon) comprises a family of extracellular signaling proteins with antiviral, antiproliferating and immunomodulatory activities. They are produced by peripheral blood leukocytes, lymphoblastoid and myeloblastoid cell lines on viral activation [2]. The interest in this protein is connected with its therapeutic value against certain types of tumors such as brain tumors and malignant melanomas [3]. Recombinant IFN-α is also used for the treatment of AIDS-related Kaposi's sarcoma and chronic hepatitis B and C [4]. Because of their increasing interest in the medical, scientific and industrial area, separation and purification of human interferons have been studied using various methods, including dye affinity chromatography, metal chelation, precipitation, ion-exchange chromatography, gel-filtration chromatography, hydrophobic interaction chromatography and immunoaffinity chromatography [5–9]. Generally, isolation and purification of proteins are very important and purified proteins are extensively used as foaming, emulsifying and gelling agent in food industry, therapeutic row material in drug industry, medical diagnostic chemicals and industrial products such
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as hair-care solutions and photography supplies due to their predisposition for surface activity [10]. The purity of a protein is a prerequisite for the studies related to its structure and function studies or its potential application. Hence, proteins should be purified before any treatment. Protein separation and purification have a high complexity. But, a wide variety of protein purification techniques are available today, however, different types of chromatography have become dominant due to their high resolving power [11]. Among the numerous techniques, protein purification via adsorption is a widely investigated phenomenon having its origin in several different disciplines of science [12]. Also, adsorption of proteins onto a polymeric solid support is the most general, the easiest to perform and the oldest protocol of physico-chemical methods and may have a higher commercial potential than other methods [13]. In the various chromatographic techniques mentioned above, separation of a protein is dependent on its biological and physicochemical properties: molecular size, net charge and other biospecific characteristics [14,15]. Protein adsorption techniques via hydrophobic interaction between protein and solid support, called hydrophobic interaction chromatography (HIC), can be favorable in many cases. Some polymeric beads can be effectively applied for HIC. HIC takes advantage of the hydrophobicity of proteins by promoting its separation on the basis of hydrophobic interactions between immobilized hydrophobic ligands and nonpolar regions on the surface of the proteins [16]. In HIC, small aminoacid molecules can be used as pseudo-specific ligands of target proteins and may hold certain advantages as ligands for industrial bioaffinity separations since they are not likely to cause an immune response in case of leakage into the product [17]. In addition, in the use of aminoacids as ligand, they can be directly put into polymeric backbone without any necessity to activate the sorbent and immobilize them onto polymeric sorbent [18–26]. To gain a hydrophobic character to adsorbents, lots of different types of hydrophobic aminoacids which have side chains of nonpolar amino acids such as alanine, methionine, tryptophan and phenyalanine on their surface can be used as a ligand in HIC [27]. These pseudospecific ligands have low binding constants (10 mM − 1 to 1 μM − 1) and, consequently, belong to weak affinity ligands family. Nevertheless, they can exhibit selectivity resulting from the cumulative effects of multiple weak binding, and van der Waals interactions with fast kinetics. L -Phenylalanine has been used as a pseudospecific hydrophobic ligand for the isolation of biomolecules [28]. In this study, it was focused on the investigation of adsorption of recombinant human interferon-α (rHuIFN-α) from aqueous solution using a hydrophobic polymeric solid support. Poly(HEMA–MAPA) microbeads are proposed as a new model support in order to achieve this aim. In the first step of the study, MAPA comonomer was synthesized by reacting methacryloyl chloride with L-phenylalanine. Secondly, HEMA was directly copolymerized with MAPA using suspension polymerization technique in order to produce poly(HEMA–MAPA) microbeads. This approach in which MAPA acts as both comonomer and specific ligand has several advantages over conventional methods. Coupling of a ligand to the adsorption matrix is an expensive, time consuming and critical step in the preparation of affinity adsorbent. In our approach, comonomer MAPA acted as ligand and it is possible to load it directly into polymeric chain of the microbeads without further activation and ligand immobilization steps. Then, the microbeads were characterized and their protein adsorption capacities and reusabilities were investigated as a function of external stimuli such as contact time, pH, ionic strength and temperature. 2. Experimental 2.1. Materials Recombinant human interferon-α (rHuIFN-α) (freeze-dried powder; 99% pure by RP-HPLC) was obtained from ProSpec-Tany
TechnoGene Ltd. (USA). L-phenylalanine and methacryloyl chloride were purchased from Sigma Chemicals Co. (St. Louis, MO). Hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EDMA) and benzoyl peroxide were obtained from Fluka A.G. (Buchs, Switzerland). HEMA was distilled under reduced pressure in the presence of hydroquinone and stored at 4 °C until use. Poly(vinyl alcohol) (PVAL; Mw: 100.000, 98% hydrolyzed) was supplied from Aldrich Chemicals Company (Milwaukee, USA). All other chemicals were of reagent grade and purchased from Merck A.G. (Darmstadt, Germany). All the water used in the experiments was purified using a Barnstead (Dubuque, IA, USA) ROpure LP ® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure ® organic/colloid removal and ion-exchange packed-bed system. 2.2. Methods 2.2.1. Synthesis of N-methacryloyl-L-phenylalanine (MAPA) monomer Applied procedure for synthesizing the functional comonomer, Nmethacryloyl-L-phenylalanine (MAPA), was reported elsewhere [11,29]. It can be given as briefly; L-phenylalanine (5.0 g) and NaNO2, used as hydrogen acceptor to promote the reaction [30–32], (0.2 g) were dissolved in 30 ml of K2CO3 aqueous solution (5%, w/v). The solution was cooled to 0 °C. Methacryloyl chloride (4.0 ml) was slowly poured into this solution under nitrogen atmosphere. This solution was then stirred magnetically at room temperature for 2 h. At the end of this chemical reaction period, the pH of the solution was exactly adjusted to 7.0 and subsequently the solution was extracted with ethyl acetate. The aqueous phase was evaporated in a rotary evaporator. The residue (i.e., MAPA) was crystallized from ether and cyclohexane. The MAPA was characterized by 1H-NMR measurement. The determined characteristic peaks are as follows: [ 1H NMR (CDCl3)] 2.84 (t, 3H, CH3), 3.05–3.19 (m, 2H, CH2), 4.80–4.85 (m, 1 H, methyne), 5.24 (s, 1H, vinyl H), 5.56 (s, 1H, vinyl), 6.24 (σ, 1H, NH), 7.04–7.20 (m, 5H, aromatic), 10.07 (s, 1H, OH). 2.2.2. Preparation of polymeric microbeads HEMA and MAPA monomers were copolymerized in suspension copolymerization by using benzoyl peroxide and poly(vinyl alcohol) as the initiator and the stabilizer, respectively. Toluene and ethylene glycol dimethacrylate (EDMA) were included in the recipe as the diluent (as a porogen) and cross-linker, respectively. A typical preparation procedure is as follows. The continuous suspension medium was prepared by dissolving poly(vinyl) alcohol (200 mg) in the purified water (50 ml). For the preparation of monomer phase, HEMA (4.0 ml), EDMA (8.0 ml) and toluene (12.0 ml) were mixed together and then MAPA (1.0 g) and benzoyl peroxide (100 mg) were dissolved in the resulting homogeneous organic monomer phase. The organic phase was dispersed in the aqueous medium by stirring the mixture magnetically (300 rpm) in a sealed cylindrical pyrex polymerization reactor. The volumetric ration between organic and aqueous was kept around 1:2 (organic: aqueous). The contents were heated to the polymerization temperature (i.e. 65 °C) within 4 h and; then, the polymerization was conducted for 2 h with a 600 rpm stirring rate at 90 °C. The plain, poly(HEMA), microbeads were prepared using the same polymerization recipe given above but without MAPA. Final microbeads were extensively washed with ethyl alcohol and water to remove any unreacted monomer or diluents. The washing steps were applied as following sequences; thrice with absolute ethyl alcohol (25 mL), thrice ethyl alcohol–water mixture (75:25; v:v), thrice ethyl alcohol–water mixture (50:50; v:v), thrice ethyl alcohol–water mixture (25:75; v:v), and then thrice deionized water. After all, the microbeads were stored in distilled water at 4 °C.
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2.2.3. Characterization of microbeads In order to determine the chemical and physical properties of the polymeric microbeads, various techniques as mentioned particularly in our studies have been used such as elemental analysis, FTIR, swelling test, and SEM micrographs [33–35]. The average size and size distribution of the poly(HEMA–MAPA) microbeads were determined by screen analysis performed using Tyler standard sieves. Water uptake ratios of the poly(HEMA–MAPA) microbeads were determined in distilled water. The experiment was conducted as follows: initially dry microbeads were carefully weighed before being placed in a 50-mL vial containing distilled water. The vial was put into an isothermal water bath with a fixed temperature (25 ± 0.5 °C) for 2 h. The microbead sample was taken out of the water, wiped using a filter paper, and weighed. The weighed ratio of dry and wet samples was recorded. The water content of the poly(HEMA–MAPA) microbeads was calculated by using the following expression: Water uptake ratio % ¼ ½ðWs −Wo Þ=Wo 100
ð1Þ
Here, Wo and Ws are the weights (g) of microbeads before and after uptake of water, respectively. The surface morphology of the microbeads was examined using scanning electron microscopy (SEM). The samples were initially dried in air at 25 °C for 7 days before being analyzed. A fragment of the dried microbead was mounted on a SEM sample mount and was sputter coated for 2 min. The sample was then mounted in a scanning electron microscope (FEI, Quanta 200 FEG, Oregon, USA). The surface of the sample was then scanned at the desired magnification to study the morphology of the poly(HEMA–MAPA) microbeads. To evaluate the degree of MAPA incorporation, the synthesized poly(HEMA– MAPA) microbeads were subjected to elemental analysis using a Leco Elemental Analyzer (model CHNS-932). The characteristic functional groups of the poly(HEMA–MAPA) microbeads were analyzed by using a Fourier transform infrared spectrophotometer (FTIR, 8000 Series, Shimadzu, Japan). The samples were prepared by mixing microbeads (2 mg) with powdered KBr (98 mg, IR Grade, Merck, Germany). Then, the mixture was pressed into a pellet form. The specific surface areas of the microbeads were determined by multipoint Brunauer–Emmett–Teller (BET) apparatus (Quantachrome, Nova 2200E, USA). Prior to measurements, the microbeads were dried in air at room temperature for one week. Dry microbead samples (1.0 g) were placed in a sample holder and degassed in a N2-gas stream at 180 °C for 1 h. Adsorption of the gas was performed at −210 °C and desorption was performed at room temperature. Data obtained from desorption step was used for the specific surface area calculation. 2.2.4. Recombinant human interferon-α adsorption and elution studies Adsorption and elution studies of rHuIFN-α were performed in batch system. rHuIFN-α adsorption onto the poly(HEMA-MAPA) microbeads was studied at various pH values, either in acetate buffer (0.1 M, pH 3.0–5.5) or in phosphate buffer (0.1 M, pH 6.0–8.0). Initial rHuIFN-α concentrations were varied in the range of 0.2–6.0 mg/ml. The adsorption experiments were conducted for 2 h at 25 °C while stirring continuously. At the end of this period, the rHuIFN-α adsorbed microbeads were removed from the solution and, then, washed with the same buffer three times. The microbeads were stored at 4 °C in fresh buffer until use. Initial and final concentrations of rHuIFN-α were measured and calculated by means of an UV–Vis spectrophotometer (Shimadzu, Model 1601, Tokyo, Japan) at 280 nm. The amount of adsorbed rHuIFN-α was calculated according to mass balance. rHuIFN-α elution studies were performed in a solution containing 0.1 M ethylene glycol. The rHuIFN-α adsorbed microbeads were placed in the elution medium and stirred continuously (150 rpm) for 1 h at 25 °C. The elution ratio was calculated from the difference between the amount of rHuIFN-α adsorbed on the microbeads and
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the amount of rHuIFN-α desorbed into the elution medium. This adsorption–elution cycle was repeated 10 times by using the same microbeads in order to obtain the reusability of them. In order to determine the effect of adsorption–desorption processes on rHuIFN-α structure, fluorescence spectrophotometer (Shimadzu, RF53010, Tokyo, Japan) was also used. The emission spectra were recorded in a range of 300–450 nm when excitations were applied at 350 nm, respectively. Other experimental parameters were applied as slit width was 5.0 nm for both excitation and emission, scan speed was super, sensitivity was high and response time and shutter were automatically controlled. 3. Results and discussion 3.1. Characterization of poly(HEMA–MAPA) microbeads In this study, it was targeted to prepare a new hydrophobic adsorbent for the affinity adsorption of rHuIFN-α from aqueous solutions in a batch system. The hydrophobic poly(HEMA–MAPA) microbeads were synthesized for this purpose. The molecular formula of the synthesized microbeads is illustrated in Fig. 1. Hydrophobic phenylalanine functional group can easily interact with the residues of rHuIFN-α protein. It should be especially noted that the affinity interactions of rHuIFN-α protein with hydrophobic MAPA groups on the adsorbent is strongly depend on the changes in both adsorbent and protein surface topography, chemical and physical variable parameters. For this reason, the poly(HEMA–MAPA) microbeads were characterized for determination of water uptake capability, surface morphology, amount of MAPA incorporation, and FTIR. Cross-linked poly(HEMA– MAPA) microbeads are insoluble in water and have sufficient mechanical and thermal stability. They do not dissolve, but can swell and protect their three-dimensional shape in aqueous solutions. The equilibrium water content of the microbeads was found to be 53.3%. Water uptake capacity of poly(HEMA–MAPA) microbeads is lower than that of poly(HEMA) microbeads (69.3%). Copolymerizing MAPA with HEMA can be considered as a possible reason; because, it effectively increased the hydrophobicity of the polymeric adsorbent. It causes a decrease in the penetration efficiency of the water molecules into polymeric chain. Therefore, MAPA incorporation decreased the water uptake capability of the microbeads. An elemental analysis depended on nitrogen stoichiometry of the poly(HEMA–MAPA) microbeads, with an average particle size distribution around 150–250 μm, were performed to evaluate the MAPA incorporation degree into polymeric network. The MAPA content of the poly(HEMA–MAPA) microbeads was found to be 327 μmol/g microbead. The specific surface area and average pore diameters determined by BET apparatus were 17.4 m 2/g and 47.3 Å for poly(HEMA) microbeads and 18.7 m 2/g and 49.8 Å for poly(HEMA–MAPA) microbeads. The results verify that MAPA incorporation causes an increase in specific surface area of microbeads. Here, it also should be noted the average pore diameters of the beads are enough for interferon molecules, the size of the protein molecules is about 3 nm in diameter [36], penetrating into pores and interacting with functional groups of the MAPA. SEM micrographs, present surface morphology of the poly(HEMA– MAPA) microbeads. Fig. 2a shows that the microbeads are spherical and have a rough surface, which is concordant with BET analysis, due to the pores that formed during the polymerization procedure. The roughness of the surface and pores having varying dimensions should be considered as a factor providing an increase in the surface area. In addition, these pores reduce diffusional resistance and facilitate mass transfer due to high internal surface area and easy diffusion to interior of the microbeads increases. This factor also enabled higher rHuIFN-α adsorption capacity. FTIR spectra of poly(HEMA–MAPA) and poly(HEMA) microbeads were undertaken to determine the chemical structure of the
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Fig. 1. (a) The molecular formula of the poly(HEMA–MAPA) microbeads and (b) proposed interaction mechanism between microbeads and rHuIFN-α molecules.
microbeads and to demonstrate the incorporation of MAPA into polymeric structure. As shown in Fig. 3, FTIR spectra of both poly(HEMA) and poly(HEMA–MAPA) microbeads have the characteristic stretching vibration band of –OH groups at 3443 cm− 1 and 3435 cm− 1, respectively. In addition, carbonyl groups can easily be seen in both spectra around 1732 cm− 1. Distinctively, poly(HEMA–MAPA) microbeads have characteristic bands originated from amide groups of MAPA monomer at 1658 cm− 1 and 1540 cm− 1, respectively. The band at 1076 cm− 1 was stemmed from benzene ring of the MAPA. Also, the bands at 1023 cm− 1 and 851 cm− 1 (rocking band) established from presence of –NH group. FTIR results show that MAPA incorporation into polymeric chain was achieved. 3.2. rHuIFN-α adsorption and elution studies from aqueous solutions 3.2.1. Adsorption kinetics Adsorption of rHuIFN-α onto poly(HEMA–MAPA) microbeads seemed to be strongly affected by the several factors which were evaluated as contact time, pH, salt type, concentration, and
temperature. MAPA was incorporated into polymeric chain and used as the rHuIFN-α binding agent. To observe the effect of contact time on rHuIFN-α adsorption onto microbeads, the samples were taken from adsorption medium at different time intervals between 5 and 120 min. The microbeads were incubated with the rHuIFN-α solution samples (2 mg/ml) at room temperature. Fig. 4 gives the equilibrium adsorption time curves that were obtained by following the decrease of the concentration of rHuIFN-α within the samples with time. As indicated here, there were relatively faster adsorption rates observed at the beginning, and then adsorption equilibria were achieved in about 30 min. Notice that there was a very low nonspecific rHuIFN-α adsorption (4.1 mg rHuIFN-α/g polymer) due to the fact that there are no reactive functional groups which interact with rHuIFN-α molecules onto the unmodified microbeads. Hence, the adsorption may be stemmed from the diffusion of rHuIFN-α into the pores and weak interactions between rHuIFN-α molecules and the microbeads. On the other hand, much higher adsorption capacities (137.6 mg rHuIFN-α/g polymer) were obtained when the poly(HEMA–MAPA) microbeads were used. The rapid adsorption
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3.2.3. Effect of pH on rHuIFN-α adsorption The pH-dependency of rHuIFN-α adsorption onto the microbeads was studied in a pH range of 3.0–8.0 (Fig. 6). In all investigated cases, the maximum adsorption was obtained at pH 6.0. At pH values lower and higher than that the adsorbed amount of rHuIFN-α decreased drastically. This may be due to both specific interactions resulted from hydrophobic MAPA ligand on adsorbent and non-polar residues of the rHuIFN-α and an increase in the conformational size and lateral electrostatic repulsions between adjacent rHuIFN-α molecules. Note that the isoelectric point of a rHuIFN-α is 5.9. The results taken from this study were convenient with theoretical previsions that the maximum adsorption of proteins from aqueous solutions is usually observed at their isoelectric points because of the fact that they have no net charge at this point [37].
Fig. 2. SEM micrographs of poly(HEMA–MAPA) microbeads: (a) surface; (b) cross section.
kinetics show both high affinity between rHUIFN-α molecules and poly(HEMA–MAPA) microbeads and the rHUIFN-α molecules did not encounter any diffusion limitation [37].
3.2.2. Effect of initial concentration of rHuIFN-α on adsorption Adsorption behavior dependency on the initial concentration was investigated by changing the concentration of the rHuIFN-α solution in a range of 0.2–6.0 mg/ml (Fig. 5). As presented in the figure, from starting concentration (0.2 mg/ml), adsorption capacity showed a significant increase, which was indicative of high affinity between the rHuIFN-α molecules and adsorbent. The high rHuIFN-α concentration may also contribute to this high adsorption capacity due to the high driving force between the solution and poly(HEMA–MAPA) solid phases. Thus, rHuIFN-α adsorption is favored at a higher initial concentration. After the concentration of 3.0 mg/ml, it became nearly constant and the plateau value of rHuIFN-α was attained by approaching saturation. This phenomenon means that all the binding sites, available for the rHuIFN-α adsorption, on the poly(HEMA– MAPA) microbead surface have been almost occupied. It should be reported that a negligible amount of rHuIFN-α was non-specifically adsorbed onto poly(HEMA) microbeads. Compared with poly(HEMA) microbeads, hydrophobic MAPA molecules significantly increased the adsorption capacity of the poly(HEMA–MAPA) microbeads. It is clear that this increase is due to specific interactions between MAPA and rHuIFN-α molecule.
3.2.4. Effect of temperature on rHuIFN-α adsorption The influence of temperature on rHuIFN-α adsorption was studied at various temperatures (5–40 °C) in order to determine the origin of the most effective interaction force between rHuIFN-α and poly (HEMA–MAPA) microbeads (Fig. 7). As seen in figure, adsorption capacity significantly ascended with the increase of temperature. It can be suggested that adsorption process was sensitive to incubation temperature in the presence of poly(HEMA–MAPA) microbeads. The non-specific adsorption of rHuIFN-α was very low and can be negligible at all temperatures. It means that no significant effect of the temperature was observed on the physical adsorption of the rHuIFN-α onto poly(HEMA) microbeads. On the other hand, the equilibrium adsorption of rHuIFN-α onto the poly(HEMA–MAPA) microbeads increased significantly with increasing temperature. This is due to the temperature responsive interaction between the hydrophobic MAPA groups and the rHuIFN-α molecules as the temperature increased. The fact may be explained by different ways [11,19,21]. First, the most effective interaction forces controlling adsorption process is hydrophobic [19]. Because, at higher temperatures protein molecules tend to unfold; so, hydrophobic residues buried into inner part of the protein molecules get out of the surface of the protein molecules. By this process, the contacting surface area of the protein molecules enlarged. That also causes an increase in affinity between protein molecules and the adsorbent [21]. Second, Van der Waals forces which are important interactions in hydrophobic chromatography, also increased by increasing temperature [11]. As conclusion, the interaction between rHUIFN-α molecules and poly(HEMA–MAPA) microbeads are mainly hydrophobic and could be promoted by increasing temperature. 3.2.5. Influence of salt type on rHuIFN-α adsorption It is well known that the salt type and concentration are very determinative parameters for protein adsorption because the interactions of proteins with solid supports are generally dependent on the ionic strength of the surrounding medium. The influence of different salts on hydrophobic interactions follows the Hofmeister (lyotropic) series for the precipitation of proteins from aqueous solutions. The salts at the beginning of these series promote hydrophobic interactions and protein precipitation (salting-out effect) and are called anti-chaotropic. They are considered to be water structuring. The addition of various structure-forming “salting out” salts to the equilibration buffer and protein solution promotes ligand–protein interactions in HIC. As the concentration of such salts is increased, the amount of proteins bound also increases significantly up to specific salt concentration. The salts at the end of the series (salting in or chaotropic ions) randomize the structure of the liquid water and thus tend to decrease the strength of hydrophobic interactions. Theoretically, the use of high concentration of anti-chaotropic salts on the equilibrium buffer and sample solution promotes the ligand–protein interactions and consequently the protein retention in HIC. The amount of the bound protein increases almost linearly with the enhancement ionic
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Fig. 3. FTIR spectra of the microbeads.
strength and continues to increase in an exponential manner at still higher concentration [38–40]. Experimental results were convenient with these facts. The effects of salt type and concentration have been studied using various salts (ammonium sulfate, (NH4)2SO4, sodium chloride, NaCl, and sodium sulfate, Na2SO4) and the results were presented in Fig. 8. The rHuIFN-α adsorption onto poly(HEMA– MAPA) microbeads ascended with the increasing concentration of anti-chaotropic NaCl and Na2SO4 salts and the maximum adsorption was achieved when Na2SO4 was used (42.9 mg/g at 0.5 mg/ml). But conversely, (NH4)2SO4 salt has chaotropic character and caused the decreasing of the rHuIFN-α adsorption.
3.2.6. Repeated use Especially from the biotechnological and commercial points of view, repeated usability provides a unique property and is one of the most important strongpoint of supports. The supports having this capability are considered as having a great influence on their extended applications in improving the process economy. In the last step of the affinity adsorption, the main concern was to elute the adsorbed protein in the shortest time and at the highest amount possible. For the desorption of rHuIFN-α, the rHuIFN-α adsorbed poly(HEMA–MAPA) microbeads were placed within the desorption medium containing 25 ml 0.1 M ethylene glycol solution at room
Fig. 4. The effect of contact time on rHuIFN-α adsorption onto poly(HEMA–MAPA) microbeads. rHuIFN-α concentration: 2 mg/ml; pH: 6.0; T: 25 °C.
Fig. 5. Effect of initial concentration on rHuIFN-α adsorption onto poly(HEMA–MAPA) microbeads. pH: 6.0; T: 25 °C.
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Fig. 6. Effect of pH on rHuIFN-α adsorption onto poly(HEMA–MAPA) microbeads. rHuIFN-α concentration: 2 mg/ml; T: 25 °C.
Fig. 8. Effect of salt concentration on rHuIFN-α adsorption onto poly(HEMA–MAPA) microbeads. rHuIFN-α concentration: 0.5 mg/ml; pH 6.0; T: 25 °C.
temperature for 1 h (Fig. 9). There was no noticeable loss in the adsorption capacity of the microbeads. After 10 cycles, the capacity had only decreased by approximately 10%. The results showed that ethylene glycol is a suitable elution agent allows repeated use of poly(HEMA– MAPA) microbeads designed in this study. This is an important feature indicating the possibility of recycling microbeads in the protein purification process. Moreover, fluorescence spectrophotometry was employed to evaluate the effects of adsorption conditions on rHuIFNα structure (Fig. 10). The fluorescence spectrum of rHuIFN-α samples obtained from the elution step was recorded. The fluorescence spectra of native and heat denaturated rHuIFN-α were also taken. A clear difference was observed between the fluorescence spectra of native rHuIFNα and heat denaturated rHuIFN-α. An appreciable shift was seen in the maximum wavelength of denaturated rHuIFN-α according to the native one. On the other hand, the fluorescence spectrum of the samples withdrawn from the elution step were very close to those of native rHuIFNα and no significant shift of maximum wavelength was detected in the spectra of these samples relative to that of native rHuIFN-α. It may be concluded that hydrophobic poly(HEMA–MAPA) microbeads can be applied for rHuIFN-α separation without causing any conformational changes and denaturation problem.
4. Conclusion Hydrophobic interaction chromatography enables one to use hydrophobic polymeric microbeads for rapid, cost-effective, and selective protein adsorption [22]. HIC may eventually constitute a significant step in purification of human interferons due to the fact that they may be a unique hydrophobic molecule as evidenced by their interaction with immobilized serum albumin, concanavalin A and the hydrocarbon side chains of substituted agarose. The main criterion for the selection of hydrophobic interaction chromatography to separate interferon molecules depends on highly hydrophobic nature of human interferons [41]. Here, we have reported hydrophobic amino acid, L-phenyl alanine, incorporated poly(HEMA–MAPA) microbeads and evaluation of their adsorptive properties for rHuIFN-α molecules. Recombinant HuIFN-α adsorption capacity of poly(HEMA–MAPA) microbeads are strongly depended on initial protein concentration, pH, temperature, salt type and contact time. After ten operational cycles, it was observed that the poly(HEMA–MAPA) microbeads retained lots of their adsorption capacity. Besides poly(HEMA–MAPA) microbeads provided an efficient separation method for rHuIFN-α with high binding capacity via hydrophobic
Fig. 7. Effect of temperature on rHuIFN-α adsorption onto poly(HEMA–MAPA) microbeads. rHuIFN-α concentration: 2 mg/ml; pH: 6.0.
Fig. 9. Repeated use of poly(HEMA–MAPA) microbeads. rHuIFN-α concentration: 2 mg/ml; pH 6.0; T: 25 °C.
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Fig. 10. Fluorescence emission spectra of recombinant, desorbed and denaturated human interferon samples. Slit width: 5.0 nm (for both excitation and emission); scan speed: super; sensitivity: high.
interaction, recovery was assured about 90%. To observe the effects of adsorption conditions on conformational changes of rHuIFN-α molecules, fluorescence spectrophotometry was employed. It appears that the poly(HEMA–MAPA) microbeads can be applied for the rHuIFN-α adsorption without causing any denaturation problem. In conclusion, the hydrophobic poly(HEMA–MAPA) microbeads can be used as an alternative adsorbent for hydrophobic rHuIFN-α purification. References [1] G.C. Sen, P. Lengyel, J. Biol. Chem. 267 (1992) 5017–5020. [2] T.A. Nyman, H. Tölö, J. Parkkinen, N. Kalkkinen, Biochem. J. 329 (1998) 295–302. [3] S. Pestka, J.A. Langer, K.C. Zoon, C.E. Samuel, Annu. Rev. Biochem. 56 (1987) 727–777. [4] S. Swaminathan, N. Khanna, Protein Expr. Purif. 15 (1999) 236–242. [5] V. Karakoç, H. Yavuz, A. Denizli, Colloids Surf. A 240 (2004) 93–99. [6] E.B. Altıntaş, A. Denizli, J. Appl. Polym. Sci. 103 (2007) 975–981. [7] L. Scapol, P. Rappuoli, G.C. Viscomi, J. Chromatogr. A 600 (1992) 235–242. [8] D. Novick, Z. Eshar, O. Gigi, Z. Marks, M. Revel, M.M. Rubinstein, J. Gen. Virol. 64 (1983) 905–910. [9] Y. Kagawa, S. Takasaki, J. Utsumi, K. Hosoi, H. Shimizu, N. Kochibe, A. Kobata, J. Biol. Chem. 263 (1988) 17508–17515. [10] S. Magdasssi, A. Kamyshny, in: S. Magdassi (Ed.), Surface Activity of Proteins: Chemical and Physicochemical Modifications, Marcell Dekker, New York, 1996, pp. 1–4.
[11] S. Oncel, L. Uzun, B. Garipcan, A. Denizli, Ind. Eng. Chem. Res. 44 (2005) 7049–7056. [12] S.H. Mollmann, J.T. Bukrinsky, S. Frokjaer, U. Elofsson, J. Colloid Interface Sci. 286 (2005) 28–35. [13] P.C. Oliveira, G.M. Alves, H.F. de Castro, Biochem. Eng. J. 5 (2000) 63–71. [14] L. Uzun, H. Yavuz, R. Say, A. Ersöz, A. Denizli, Ind. Eng. Chem. Res. 43 (2004) 6507–6513. [15] H. Yavuz, A. Denizli, Macromol. Biosci. 4 (2004) 84–91. [16] J.L. Fausnaugh, F.E. Regnier, J. Chromatogr. 359 (1986) 131–146. [17] N. Bereli, L. Uzun, H. Yavuz, A. Elkak, A. Denizli, J. Appl. Polym. Sci. 101 (2006) 395–404. [18] G. Sener, E. Ozgur, E. Yilmaz, L. Uzun, R. Say, A. Denizli, Biosens. Bioelectron. 26 (2010) 815–821. [19] L. Uzun, R. Say, S. Unal, A. Denizli, J. Chromatogr. B 877 (2009) 181–188. [20] L. Uzun, R. Say, S. Unal, A. Denizli, Biosens. Bioelectron. 24 (2009) 2878–2884. [21] E. Yılmaz, L. Uzun, A.Y. Rad, U. Kalyoncu, S. Unal, A. Denizli, J. Biomater. Sci. Polym. Ed. 19 (2008) 875–892. [22] G. Ertürk, L. Uzun, M.A. Tümer, R. Say, A. Denizli, Biosens. Bioelectron. 28 (2011) 97–104. [23] S. Aslıyüce, N. Bereli, L. Uzun, M.A. Onur, R. Say, A. Denizli, Sep. Purif. Technol. 73 (2010) 243–249. [24] L. Uzun, H. Yavuz, B. Osman, H. Çelik, A. Denizli, Int. J. Biol. Macromol. 47 (2010) 44–49. [25] B. Akkaya, L. Uzun, F. Candan, A. Denizli, Mater. Sci. Eng. C 27 (2007) 180–187. [26] N. Bereli, Y. Saylan, L. Uzun, R. Say, A. Denizli, Sep. Purif. Technol. 82 (2011) 28–35. [27] O. Pitiot, C. Legallais, L. Darnige, M.A. Vijayalakshmi, J. Membr. Sci. 166 (2000) 221–227. [28] M. Kim, K. Saito, S. Furusaki, T. Sato, T. Sugo, T.I. Ishigaki, J. Chromatogr. 585 (1991) 45–51. [29] M.Y. Arica, Y. Kacar, A. Ergene, A. Denizli, Process Biochem. 36 (8–9) (2001) 847–854. [30] M.B. Smith, J. March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley-Interscience, New York, USA, 2001. [31] C. Schotten, Ber. Dtsch. Chem. Ges. 17 (1884) 2544. [32] E. Baumann, Ber. Dtsch. Chem. Ges. 19 (1886) 3218. [33] L. Uzun, A. Kara, N. Tüzmen, A. Karabakan, N. Beşirli, A. Denizli, J. Appl. Polym. Sci. 102 (2006) 4276–4283. [34] E.B. Altıntaş, L. Uzun, A. Denizli, China Part. 5 (2007) 174–179. [35] N. Başar, L. Uzun, A. Güner, A. Denizli, J. Appl. Polym. Sci. 108 (2008) 3454–3461. [36] M.S. Schwarzenbach, P. Reimann, V. Thommen, M. Hegner, M. Mumenthaler, J. Schwob, H.J. Güntherodt, J. Pharm. Sci. Technol. 56 (2002) 78–89. [37] M.M. Sari, C. Armutcu, N. Bereli, L. Uzun, A. Denizli, Colloids Surf. B 84 (2011) 140–147. [38] S. Roe, in: E.L.V. Harris, S. Angal (Eds.), Oxford, IRL Press, New York, 1989, pp. 221–232. [39] J. Porath, Biotechnol. Progr. 3 (1987) 14–21. [40] J.A. Queiroz, F.A.P. Garcia, J.M.S. Cabral, J. Chromatogr. A 734 (1996) 213–219. [41] M.W. Davey, J.W. Huang, E. Sulkowski, W.A. Carter, J. Biol. Chem. 250 (1975) 348–349.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Hydrophobic microbeads as an alternative pseudo-affinity adsorbent for recombinant human interferon-α via hydrophobic interactions Yeşeren Saylan a, Müfrettin Murat Sarı b,⁎, Serpil Özkara c, d, Lokman Uzun a,⁎, Adil Denizli a a
Hacettepe University, Department of Chemistry, Biochemistry Division, Ankara, Turkey Turkish Military Academy, Department of Basic Sciences, Ankara, Turkey Inonu University, Faculty of Pharmacy, Biochemistry Division, Malatya, Turkey d Anadolu University, Department of Chemistry, Biochemistry Division, Eskişehir, Turkey b c
a r t i c l e
i n f o
Article history: Received 15 January 2011 Received in revised form 15 November 2011 Accepted 2 February 2012 Available online 9 February 2012 Keywords: Recombinant human interferon-α Hydrophobic interaction chromatography Phenylalanine Poly(HEMA–MAPA) microbeads Affinity adsorption Pseudo-affinity adsorbents
a b s t r a c t Hydrophobic interaction chromatography (HIC) is increasingly used for protein purification, separation and other biochemical applications. The aim of this study was to prepare hydrophobic microbeads and to investigate their recombinant human interferon-α (rHuIFN-α) adsorption capability. For this purpose, we had synthesized functional monomer, N-methacryloyl-L-phenylalanine (MAPA), to provide a hydrophobic functionality to the adsorbent. The poly(2-hydroxyethyl methacrylate-N-methacryloyl-L-phenylalanine) [poly(HEMA–MAPA)] microbeads were prepared by suspension copolymerization. microbeads were characterized using FTIR, swelling behavior, and SEM micrographs. Equilibrium swelling ratio of poly(HEMA– MAPA) and poly(HEMA) microbeads were 53.3% and 69.3%, respectively. The specific surface area and average pore diameters determined by BET apparatus were 17.4 m2/g and 47.3 Å for poly(HEMA) microbeads and 18.7 m2/g and 49.8 Å for poly(HEMA–MAPA) microbeads. Adsorption experiments were performed under different conditions. Maximum rHuIFN-α adsorption capacity was found to be 137.6 ± 6.7 mg/g by using poly(HEMA–MAPA) microbeads with a size range of 150–250 μm and containing 327 μmol MAPA/g microbeads. Compared with poly(HEMA–MAPA) microbeads, nonspecific rHuIFN-α adsorption onto plain poly(HEMA) microbeads was very low, about 4.2 ± 2.3 mg/g. To determine the effects of adsorption conditions on possible conformational changes of rHuIFN-α structure, fluorescence spectrophotometry was employed. Repeated adsorption–elution processes showed that these microbeads are suitable for repeatable rHuIFN-α adsorption. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Interferons, named for its ability to interfere with viral proliferation, are very important proteins for the immune system and are the members of large class of glycoproteins known as cytokines. In some cases, they modulate specific cellular functions but the main function of these proteins is playing an important role in resistance to infection especially in the first line of defense against viral infections. They are part of the non-specific immune system and are induced at an early stage in viral infection before the specific immune system has had time to respond. Interferons show their main function as preventing viral replication in newly infected cells. These potent biologically active proteins are most rapidly produced by somatic cells in response to an appropriate stimulus in the presence of pathogens such as viruses, bacteria, or parasites, or other antigens or tumor cells. Then, they are secreted into the surrounding medium, bind to receptor on target cells and induce transcription of some genes and ⁎ Corresponding authors. Tel.: + 90 312 417 5190; fax: + 90 312 418 3226. E-mail addresses: [email protected] (M.M. Sarı), [email protected] (L. Uzun). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.02.016
these results in an anti-viral state in the target cells. Since interferons have different antigenic and structural characteristics, they are typically divided into three types: alpha (α), beta (β) and gamma (γ) [1]. Human interferon-α (HuIFN-α, also known as leukocyte interferon) comprises a family of extracellular signaling proteins with antiviral, antiproliferating and immunomodulatory activities. They are produced by peripheral blood leukocytes, lymphoblastoid and myeloblastoid cell lines on viral activation [2]. The interest in this protein is connected with its therapeutic value against certain types of tumors such as brain tumors and malignant melanomas [3]. Recombinant IFN-α is also used for the treatment of AIDS-related Kaposi's sarcoma and chronic hepatitis B and C [4]. Because of their increasing interest in the medical, scientific and industrial area, separation and purification of human interferons have been studied using various methods, including dye affinity chromatography, metal chelation, precipitation, ion-exchange chromatography, gel-filtration chromatography, hydrophobic interaction chromatography and immunoaffinity chromatography [5–9]. Generally, isolation and purification of proteins are very important and purified proteins are extensively used as foaming, emulsifying and gelling agent in food industry, therapeutic row material in drug industry, medical diagnostic chemicals and industrial products such
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as hair-care solutions and photography supplies due to their predisposition for surface activity [10]. The purity of a protein is a prerequisite for the studies related to its structure and function studies or its potential application. Hence, proteins should be purified before any treatment. Protein separation and purification have a high complexity. But, a wide variety of protein purification techniques are available today, however, different types of chromatography have become dominant due to their high resolving power [11]. Among the numerous techniques, protein purification via adsorption is a widely investigated phenomenon having its origin in several different disciplines of science [12]. Also, adsorption of proteins onto a polymeric solid support is the most general, the easiest to perform and the oldest protocol of physico-chemical methods and may have a higher commercial potential than other methods [13]. In the various chromatographic techniques mentioned above, separation of a protein is dependent on its biological and physicochemical properties: molecular size, net charge and other biospecific characteristics [14,15]. Protein adsorption techniques via hydrophobic interaction between protein and solid support, called hydrophobic interaction chromatography (HIC), can be favorable in many cases. Some polymeric beads can be effectively applied for HIC. HIC takes advantage of the hydrophobicity of proteins by promoting its separation on the basis of hydrophobic interactions between immobilized hydrophobic ligands and nonpolar regions on the surface of the proteins [16]. In HIC, small aminoacid molecules can be used as pseudo-specific ligands of target proteins and may hold certain advantages as ligands for industrial bioaffinity separations since they are not likely to cause an immune response in case of leakage into the product [17]. In addition, in the use of aminoacids as ligand, they can be directly put into polymeric backbone without any necessity to activate the sorbent and immobilize them onto polymeric sorbent [18–26]. To gain a hydrophobic character to adsorbents, lots of different types of hydrophobic aminoacids which have side chains of nonpolar amino acids such as alanine, methionine, tryptophan and phenyalanine on their surface can be used as a ligand in HIC [27]. These pseudospecific ligands have low binding constants (10 mM − 1 to 1 μM − 1) and, consequently, belong to weak affinity ligands family. Nevertheless, they can exhibit selectivity resulting from the cumulative effects of multiple weak binding, and van der Waals interactions with fast kinetics. L -Phenylalanine has been used as a pseudospecific hydrophobic ligand for the isolation of biomolecules [28]. In this study, it was focused on the investigation of adsorption of recombinant human interferon-α (rHuIFN-α) from aqueous solution using a hydrophobic polymeric solid support. Poly(HEMA–MAPA) microbeads are proposed as a new model support in order to achieve this aim. In the first step of the study, MAPA comonomer was synthesized by reacting methacryloyl chloride with L-phenylalanine. Secondly, HEMA was directly copolymerized with MAPA using suspension polymerization technique in order to produce poly(HEMA–MAPA) microbeads. This approach in which MAPA acts as both comonomer and specific ligand has several advantages over conventional methods. Coupling of a ligand to the adsorption matrix is an expensive, time consuming and critical step in the preparation of affinity adsorbent. In our approach, comonomer MAPA acted as ligand and it is possible to load it directly into polymeric chain of the microbeads without further activation and ligand immobilization steps. Then, the microbeads were characterized and their protein adsorption capacities and reusabilities were investigated as a function of external stimuli such as contact time, pH, ionic strength and temperature. 2. Experimental 2.1. Materials Recombinant human interferon-α (rHuIFN-α) (freeze-dried powder; 99% pure by RP-HPLC) was obtained from ProSpec-Tany
TechnoGene Ltd. (USA). L-phenylalanine and methacryloyl chloride were purchased from Sigma Chemicals Co. (St. Louis, MO). Hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EDMA) and benzoyl peroxide were obtained from Fluka A.G. (Buchs, Switzerland). HEMA was distilled under reduced pressure in the presence of hydroquinone and stored at 4 °C until use. Poly(vinyl alcohol) (PVAL; Mw: 100.000, 98% hydrolyzed) was supplied from Aldrich Chemicals Company (Milwaukee, USA). All other chemicals were of reagent grade and purchased from Merck A.G. (Darmstadt, Germany). All the water used in the experiments was purified using a Barnstead (Dubuque, IA, USA) ROpure LP ® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure ® organic/colloid removal and ion-exchange packed-bed system. 2.2. Methods 2.2.1. Synthesis of N-methacryloyl-L-phenylalanine (MAPA) monomer Applied procedure for synthesizing the functional comonomer, Nmethacryloyl-L-phenylalanine (MAPA), was reported elsewhere [11,29]. It can be given as briefly; L-phenylalanine (5.0 g) and NaNO2, used as hydrogen acceptor to promote the reaction [30–32], (0.2 g) were dissolved in 30 ml of K2CO3 aqueous solution (5%, w/v). The solution was cooled to 0 °C. Methacryloyl chloride (4.0 ml) was slowly poured into this solution under nitrogen atmosphere. This solution was then stirred magnetically at room temperature for 2 h. At the end of this chemical reaction period, the pH of the solution was exactly adjusted to 7.0 and subsequently the solution was extracted with ethyl acetate. The aqueous phase was evaporated in a rotary evaporator. The residue (i.e., MAPA) was crystallized from ether and cyclohexane. The MAPA was characterized by 1H-NMR measurement. The determined characteristic peaks are as follows: [ 1H NMR (CDCl3)] 2.84 (t, 3H, CH3), 3.05–3.19 (m, 2H, CH2), 4.80–4.85 (m, 1 H, methyne), 5.24 (s, 1H, vinyl H), 5.56 (s, 1H, vinyl), 6.24 (σ, 1H, NH), 7.04–7.20 (m, 5H, aromatic), 10.07 (s, 1H, OH). 2.2.2. Preparation of polymeric microbeads HEMA and MAPA monomers were copolymerized in suspension copolymerization by using benzoyl peroxide and poly(vinyl alcohol) as the initiator and the stabilizer, respectively. Toluene and ethylene glycol dimethacrylate (EDMA) were included in the recipe as the diluent (as a porogen) and cross-linker, respectively. A typical preparation procedure is as follows. The continuous suspension medium was prepared by dissolving poly(vinyl) alcohol (200 mg) in the purified water (50 ml). For the preparation of monomer phase, HEMA (4.0 ml), EDMA (8.0 ml) and toluene (12.0 ml) were mixed together and then MAPA (1.0 g) and benzoyl peroxide (100 mg) were dissolved in the resulting homogeneous organic monomer phase. The organic phase was dispersed in the aqueous medium by stirring the mixture magnetically (300 rpm) in a sealed cylindrical pyrex polymerization reactor. The volumetric ration between organic and aqueous was kept around 1:2 (organic: aqueous). The contents were heated to the polymerization temperature (i.e. 65 °C) within 4 h and; then, the polymerization was conducted for 2 h with a 600 rpm stirring rate at 90 °C. The plain, poly(HEMA), microbeads were prepared using the same polymerization recipe given above but without MAPA. Final microbeads were extensively washed with ethyl alcohol and water to remove any unreacted monomer or diluents. The washing steps were applied as following sequences; thrice with absolute ethyl alcohol (25 mL), thrice ethyl alcohol–water mixture (75:25; v:v), thrice ethyl alcohol–water mixture (50:50; v:v), thrice ethyl alcohol–water mixture (25:75; v:v), and then thrice deionized water. After all, the microbeads were stored in distilled water at 4 °C.
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2.2.3. Characterization of microbeads In order to determine the chemical and physical properties of the polymeric microbeads, various techniques as mentioned particularly in our studies have been used such as elemental analysis, FTIR, swelling test, and SEM micrographs [33–35]. The average size and size distribution of the poly(HEMA–MAPA) microbeads were determined by screen analysis performed using Tyler standard sieves. Water uptake ratios of the poly(HEMA–MAPA) microbeads were determined in distilled water. The experiment was conducted as follows: initially dry microbeads were carefully weighed before being placed in a 50-mL vial containing distilled water. The vial was put into an isothermal water bath with a fixed temperature (25 ± 0.5 °C) for 2 h. The microbead sample was taken out of the water, wiped using a filter paper, and weighed. The weighed ratio of dry and wet samples was recorded. The water content of the poly(HEMA–MAPA) microbeads was calculated by using the following expression: Water uptake ratio % ¼ ½ðWs −Wo Þ=Wo 100
ð1Þ
Here, Wo and Ws are the weights (g) of microbeads before and after uptake of water, respectively. The surface morphology of the microbeads was examined using scanning electron microscopy (SEM). The samples were initially dried in air at 25 °C for 7 days before being analyzed. A fragment of the dried microbead was mounted on a SEM sample mount and was sputter coated for 2 min. The sample was then mounted in a scanning electron microscope (FEI, Quanta 200 FEG, Oregon, USA). The surface of the sample was then scanned at the desired magnification to study the morphology of the poly(HEMA–MAPA) microbeads. To evaluate the degree of MAPA incorporation, the synthesized poly(HEMA– MAPA) microbeads were subjected to elemental analysis using a Leco Elemental Analyzer (model CHNS-932). The characteristic functional groups of the poly(HEMA–MAPA) microbeads were analyzed by using a Fourier transform infrared spectrophotometer (FTIR, 8000 Series, Shimadzu, Japan). The samples were prepared by mixing microbeads (2 mg) with powdered KBr (98 mg, IR Grade, Merck, Germany). Then, the mixture was pressed into a pellet form. The specific surface areas of the microbeads were determined by multipoint Brunauer–Emmett–Teller (BET) apparatus (Quantachrome, Nova 2200E, USA). Prior to measurements, the microbeads were dried in air at room temperature for one week. Dry microbead samples (1.0 g) were placed in a sample holder and degassed in a N2-gas stream at 180 °C for 1 h. Adsorption of the gas was performed at −210 °C and desorption was performed at room temperature. Data obtained from desorption step was used for the specific surface area calculation. 2.2.4. Recombinant human interferon-α adsorption and elution studies Adsorption and elution studies of rHuIFN-α were performed in batch system. rHuIFN-α adsorption onto the poly(HEMA-MAPA) microbeads was studied at various pH values, either in acetate buffer (0.1 M, pH 3.0–5.5) or in phosphate buffer (0.1 M, pH 6.0–8.0). Initial rHuIFN-α concentrations were varied in the range of 0.2–6.0 mg/ml. The adsorption experiments were conducted for 2 h at 25 °C while stirring continuously. At the end of this period, the rHuIFN-α adsorbed microbeads were removed from the solution and, then, washed with the same buffer three times. The microbeads were stored at 4 °C in fresh buffer until use. Initial and final concentrations of rHuIFN-α were measured and calculated by means of an UV–Vis spectrophotometer (Shimadzu, Model 1601, Tokyo, Japan) at 280 nm. The amount of adsorbed rHuIFN-α was calculated according to mass balance. rHuIFN-α elution studies were performed in a solution containing 0.1 M ethylene glycol. The rHuIFN-α adsorbed microbeads were placed in the elution medium and stirred continuously (150 rpm) for 1 h at 25 °C. The elution ratio was calculated from the difference between the amount of rHuIFN-α adsorbed on the microbeads and
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the amount of rHuIFN-α desorbed into the elution medium. This adsorption–elution cycle was repeated 10 times by using the same microbeads in order to obtain the reusability of them. In order to determine the effect of adsorption–desorption processes on rHuIFN-α structure, fluorescence spectrophotometer (Shimadzu, RF53010, Tokyo, Japan) was also used. The emission spectra were recorded in a range of 300–450 nm when excitations were applied at 350 nm, respectively. Other experimental parameters were applied as slit width was 5.0 nm for both excitation and emission, scan speed was super, sensitivity was high and response time and shutter were automatically controlled. 3. Results and discussion 3.1. Characterization of poly(HEMA–MAPA) microbeads In this study, it was targeted to prepare a new hydrophobic adsorbent for the affinity adsorption of rHuIFN-α from aqueous solutions in a batch system. The hydrophobic poly(HEMA–MAPA) microbeads were synthesized for this purpose. The molecular formula of the synthesized microbeads is illustrated in Fig. 1. Hydrophobic phenylalanine functional group can easily interact with the residues of rHuIFN-α protein. It should be especially noted that the affinity interactions of rHuIFN-α protein with hydrophobic MAPA groups on the adsorbent is strongly depend on the changes in both adsorbent and protein surface topography, chemical and physical variable parameters. For this reason, the poly(HEMA–MAPA) microbeads were characterized for determination of water uptake capability, surface morphology, amount of MAPA incorporation, and FTIR. Cross-linked poly(HEMA– MAPA) microbeads are insoluble in water and have sufficient mechanical and thermal stability. They do not dissolve, but can swell and protect their three-dimensional shape in aqueous solutions. The equilibrium water content of the microbeads was found to be 53.3%. Water uptake capacity of poly(HEMA–MAPA) microbeads is lower than that of poly(HEMA) microbeads (69.3%). Copolymerizing MAPA with HEMA can be considered as a possible reason; because, it effectively increased the hydrophobicity of the polymeric adsorbent. It causes a decrease in the penetration efficiency of the water molecules into polymeric chain. Therefore, MAPA incorporation decreased the water uptake capability of the microbeads. An elemental analysis depended on nitrogen stoichiometry of the poly(HEMA–MAPA) microbeads, with an average particle size distribution around 150–250 μm, were performed to evaluate the MAPA incorporation degree into polymeric network. The MAPA content of the poly(HEMA–MAPA) microbeads was found to be 327 μmol/g microbead. The specific surface area and average pore diameters determined by BET apparatus were 17.4 m 2/g and 47.3 Å for poly(HEMA) microbeads and 18.7 m 2/g and 49.8 Å for poly(HEMA–MAPA) microbeads. The results verify that MAPA incorporation causes an increase in specific surface area of microbeads. Here, it also should be noted the average pore diameters of the beads are enough for interferon molecules, the size of the protein molecules is about 3 nm in diameter [36], penetrating into pores and interacting with functional groups of the MAPA. SEM micrographs, present surface morphology of the poly(HEMA– MAPA) microbeads. Fig. 2a shows that the microbeads are spherical and have a rough surface, which is concordant with BET analysis, due to the pores that formed during the polymerization procedure. The roughness of the surface and pores having varying dimensions should be considered as a factor providing an increase in the surface area. In addition, these pores reduce diffusional resistance and facilitate mass transfer due to high internal surface area and easy diffusion to interior of the microbeads increases. This factor also enabled higher rHuIFN-α adsorption capacity. FTIR spectra of poly(HEMA–MAPA) and poly(HEMA) microbeads were undertaken to determine the chemical structure of the
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Fig. 1. (a) The molecular formula of the poly(HEMA–MAPA) microbeads and (b) proposed interaction mechanism between microbeads and rHuIFN-α molecules.
microbeads and to demonstrate the incorporation of MAPA into polymeric structure. As shown in Fig. 3, FTIR spectra of both poly(HEMA) and poly(HEMA–MAPA) microbeads have the characteristic stretching vibration band of –OH groups at 3443 cm− 1 and 3435 cm− 1, respectively. In addition, carbonyl groups can easily be seen in both spectra around 1732 cm− 1. Distinctively, poly(HEMA–MAPA) microbeads have characteristic bands originated from amide groups of MAPA monomer at 1658 cm− 1 and 1540 cm− 1, respectively. The band at 1076 cm− 1 was stemmed from benzene ring of the MAPA. Also, the bands at 1023 cm− 1 and 851 cm− 1 (rocking band) established from presence of –NH group. FTIR results show that MAPA incorporation into polymeric chain was achieved. 3.2. rHuIFN-α adsorption and elution studies from aqueous solutions 3.2.1. Adsorption kinetics Adsorption of rHuIFN-α onto poly(HEMA–MAPA) microbeads seemed to be strongly affected by the several factors which were evaluated as contact time, pH, salt type, concentration, and
temperature. MAPA was incorporated into polymeric chain and used as the rHuIFN-α binding agent. To observe the effect of contact time on rHuIFN-α adsorption onto microbeads, the samples were taken from adsorption medium at different time intervals between 5 and 120 min. The microbeads were incubated with the rHuIFN-α solution samples (2 mg/ml) at room temperature. Fig. 4 gives the equilibrium adsorption time curves that were obtained by following the decrease of the concentration of rHuIFN-α within the samples with time. As indicated here, there were relatively faster adsorption rates observed at the beginning, and then adsorption equilibria were achieved in about 30 min. Notice that there was a very low nonspecific rHuIFN-α adsorption (4.1 mg rHuIFN-α/g polymer) due to the fact that there are no reactive functional groups which interact with rHuIFN-α molecules onto the unmodified microbeads. Hence, the adsorption may be stemmed from the diffusion of rHuIFN-α into the pores and weak interactions between rHuIFN-α molecules and the microbeads. On the other hand, much higher adsorption capacities (137.6 mg rHuIFN-α/g polymer) were obtained when the poly(HEMA–MAPA) microbeads were used. The rapid adsorption
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3.2.3. Effect of pH on rHuIFN-α adsorption The pH-dependency of rHuIFN-α adsorption onto the microbeads was studied in a pH range of 3.0–8.0 (Fig. 6). In all investigated cases, the maximum adsorption was obtained at pH 6.0. At pH values lower and higher than that the adsorbed amount of rHuIFN-α decreased drastically. This may be due to both specific interactions resulted from hydrophobic MAPA ligand on adsorbent and non-polar residues of the rHuIFN-α and an increase in the conformational size and lateral electrostatic repulsions between adjacent rHuIFN-α molecules. Note that the isoelectric point of a rHuIFN-α is 5.9. The results taken from this study were convenient with theoretical previsions that the maximum adsorption of proteins from aqueous solutions is usually observed at their isoelectric points because of the fact that they have no net charge at this point [37].
Fig. 2. SEM micrographs of poly(HEMA–MAPA) microbeads: (a) surface; (b) cross section.
kinetics show both high affinity between rHUIFN-α molecules and poly(HEMA–MAPA) microbeads and the rHUIFN-α molecules did not encounter any diffusion limitation [37].
3.2.2. Effect of initial concentration of rHuIFN-α on adsorption Adsorption behavior dependency on the initial concentration was investigated by changing the concentration of the rHuIFN-α solution in a range of 0.2–6.0 mg/ml (Fig. 5). As presented in the figure, from starting concentration (0.2 mg/ml), adsorption capacity showed a significant increase, which was indicative of high affinity between the rHuIFN-α molecules and adsorbent. The high rHuIFN-α concentration may also contribute to this high adsorption capacity due to the high driving force between the solution and poly(HEMA–MAPA) solid phases. Thus, rHuIFN-α adsorption is favored at a higher initial concentration. After the concentration of 3.0 mg/ml, it became nearly constant and the plateau value of rHuIFN-α was attained by approaching saturation. This phenomenon means that all the binding sites, available for the rHuIFN-α adsorption, on the poly(HEMA– MAPA) microbead surface have been almost occupied. It should be reported that a negligible amount of rHuIFN-α was non-specifically adsorbed onto poly(HEMA) microbeads. Compared with poly(HEMA) microbeads, hydrophobic MAPA molecules significantly increased the adsorption capacity of the poly(HEMA–MAPA) microbeads. It is clear that this increase is due to specific interactions between MAPA and rHuIFN-α molecule.
3.2.4. Effect of temperature on rHuIFN-α adsorption The influence of temperature on rHuIFN-α adsorption was studied at various temperatures (5–40 °C) in order to determine the origin of the most effective interaction force between rHuIFN-α and poly (HEMA–MAPA) microbeads (Fig. 7). As seen in figure, adsorption capacity significantly ascended with the increase of temperature. It can be suggested that adsorption process was sensitive to incubation temperature in the presence of poly(HEMA–MAPA) microbeads. The non-specific adsorption of rHuIFN-α was very low and can be negligible at all temperatures. It means that no significant effect of the temperature was observed on the physical adsorption of the rHuIFN-α onto poly(HEMA) microbeads. On the other hand, the equilibrium adsorption of rHuIFN-α onto the poly(HEMA–MAPA) microbeads increased significantly with increasing temperature. This is due to the temperature responsive interaction between the hydrophobic MAPA groups and the rHuIFN-α molecules as the temperature increased. The fact may be explained by different ways [11,19,21]. First, the most effective interaction forces controlling adsorption process is hydrophobic [19]. Because, at higher temperatures protein molecules tend to unfold; so, hydrophobic residues buried into inner part of the protein molecules get out of the surface of the protein molecules. By this process, the contacting surface area of the protein molecules enlarged. That also causes an increase in affinity between protein molecules and the adsorbent [21]. Second, Van der Waals forces which are important interactions in hydrophobic chromatography, also increased by increasing temperature [11]. As conclusion, the interaction between rHUIFN-α molecules and poly(HEMA–MAPA) microbeads are mainly hydrophobic and could be promoted by increasing temperature. 3.2.5. Influence of salt type on rHuIFN-α adsorption It is well known that the salt type and concentration are very determinative parameters for protein adsorption because the interactions of proteins with solid supports are generally dependent on the ionic strength of the surrounding medium. The influence of different salts on hydrophobic interactions follows the Hofmeister (lyotropic) series for the precipitation of proteins from aqueous solutions. The salts at the beginning of these series promote hydrophobic interactions and protein precipitation (salting-out effect) and are called anti-chaotropic. They are considered to be water structuring. The addition of various structure-forming “salting out” salts to the equilibration buffer and protein solution promotes ligand–protein interactions in HIC. As the concentration of such salts is increased, the amount of proteins bound also increases significantly up to specific salt concentration. The salts at the end of the series (salting in or chaotropic ions) randomize the structure of the liquid water and thus tend to decrease the strength of hydrophobic interactions. Theoretically, the use of high concentration of anti-chaotropic salts on the equilibrium buffer and sample solution promotes the ligand–protein interactions and consequently the protein retention in HIC. The amount of the bound protein increases almost linearly with the enhancement ionic
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Fig. 3. FTIR spectra of the microbeads.
strength and continues to increase in an exponential manner at still higher concentration [38–40]. Experimental results were convenient with these facts. The effects of salt type and concentration have been studied using various salts (ammonium sulfate, (NH4)2SO4, sodium chloride, NaCl, and sodium sulfate, Na2SO4) and the results were presented in Fig. 8. The rHuIFN-α adsorption onto poly(HEMA– MAPA) microbeads ascended with the increasing concentration of anti-chaotropic NaCl and Na2SO4 salts and the maximum adsorption was achieved when Na2SO4 was used (42.9 mg/g at 0.5 mg/ml). But conversely, (NH4)2SO4 salt has chaotropic character and caused the decreasing of the rHuIFN-α adsorption.
3.2.6. Repeated use Especially from the biotechnological and commercial points of view, repeated usability provides a unique property and is one of the most important strongpoint of supports. The supports having this capability are considered as having a great influence on their extended applications in improving the process economy. In the last step of the affinity adsorption, the main concern was to elute the adsorbed protein in the shortest time and at the highest amount possible. For the desorption of rHuIFN-α, the rHuIFN-α adsorbed poly(HEMA–MAPA) microbeads were placed within the desorption medium containing 25 ml 0.1 M ethylene glycol solution at room
Fig. 4. The effect of contact time on rHuIFN-α adsorption onto poly(HEMA–MAPA) microbeads. rHuIFN-α concentration: 2 mg/ml; pH: 6.0; T: 25 °C.
Fig. 5. Effect of initial concentration on rHuIFN-α adsorption onto poly(HEMA–MAPA) microbeads. pH: 6.0; T: 25 °C.
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Fig. 6. Effect of pH on rHuIFN-α adsorption onto poly(HEMA–MAPA) microbeads. rHuIFN-α concentration: 2 mg/ml; T: 25 °C.
Fig. 8. Effect of salt concentration on rHuIFN-α adsorption onto poly(HEMA–MAPA) microbeads. rHuIFN-α concentration: 0.5 mg/ml; pH 6.0; T: 25 °C.
temperature for 1 h (Fig. 9). There was no noticeable loss in the adsorption capacity of the microbeads. After 10 cycles, the capacity had only decreased by approximately 10%. The results showed that ethylene glycol is a suitable elution agent allows repeated use of poly(HEMA– MAPA) microbeads designed in this study. This is an important feature indicating the possibility of recycling microbeads in the protein purification process. Moreover, fluorescence spectrophotometry was employed to evaluate the effects of adsorption conditions on rHuIFNα structure (Fig. 10). The fluorescence spectrum of rHuIFN-α samples obtained from the elution step was recorded. The fluorescence spectra of native and heat denaturated rHuIFN-α were also taken. A clear difference was observed between the fluorescence spectra of native rHuIFNα and heat denaturated rHuIFN-α. An appreciable shift was seen in the maximum wavelength of denaturated rHuIFN-α according to the native one. On the other hand, the fluorescence spectrum of the samples withdrawn from the elution step were very close to those of native rHuIFNα and no significant shift of maximum wavelength was detected in the spectra of these samples relative to that of native rHuIFN-α. It may be concluded that hydrophobic poly(HEMA–MAPA) microbeads can be applied for rHuIFN-α separation without causing any conformational changes and denaturation problem.
4. Conclusion Hydrophobic interaction chromatography enables one to use hydrophobic polymeric microbeads for rapid, cost-effective, and selective protein adsorption [22]. HIC may eventually constitute a significant step in purification of human interferons due to the fact that they may be a unique hydrophobic molecule as evidenced by their interaction with immobilized serum albumin, concanavalin A and the hydrocarbon side chains of substituted agarose. The main criterion for the selection of hydrophobic interaction chromatography to separate interferon molecules depends on highly hydrophobic nature of human interferons [41]. Here, we have reported hydrophobic amino acid, L-phenyl alanine, incorporated poly(HEMA–MAPA) microbeads and evaluation of their adsorptive properties for rHuIFN-α molecules. Recombinant HuIFN-α adsorption capacity of poly(HEMA–MAPA) microbeads are strongly depended on initial protein concentration, pH, temperature, salt type and contact time. After ten operational cycles, it was observed that the poly(HEMA–MAPA) microbeads retained lots of their adsorption capacity. Besides poly(HEMA–MAPA) microbeads provided an efficient separation method for rHuIFN-α with high binding capacity via hydrophobic
Fig. 7. Effect of temperature on rHuIFN-α adsorption onto poly(HEMA–MAPA) microbeads. rHuIFN-α concentration: 2 mg/ml; pH: 6.0.
Fig. 9. Repeated use of poly(HEMA–MAPA) microbeads. rHuIFN-α concentration: 2 mg/ml; pH 6.0; T: 25 °C.
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Fig. 10. Fluorescence emission spectra of recombinant, desorbed and denaturated human interferon samples. Slit width: 5.0 nm (for both excitation and emission); scan speed: super; sensitivity: high.
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